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Vitro Analysis (vitro + analysis)
Selected AbstractsDevelopment of Air Micro Bubbles in the Venous Outlet Line: An In Vitro Analysis of Various Air Traps Used for HemodialysisARTIFICIAL ORGANS, Issue 6 2007Christofer J. Stegmayr Abstract:, Venous air traps were tested in vitro with respect to presence of micro bubbles. Three types of venous air traps were measured (Bioline, Bioline GmbH, Luckenwalde, Germany; Gambro, Gambro AB, Lund, Sweden; Fresenius M.C., Fresenius Medical Care AG & Co. KGaA, Bad Homburg, Germany). Measurements (n = 10) were taken for each air trap, fluid flow (50,600 mL/min), and fluid level (high/low). A 1.5-MHz ultrasound probe was used with an analysis device. The probe was mounted on the outlet line downstream of the venous air trap. A semisynthetic fluid was used to resemble blood viscosity. Occurrences of micro bubbles, without inducing an alarm of the dialysis device, were detected in almost all measurements. The amount of bubbles increased with increasing flow. There were more bubbles with low fluid level compared with high level. The Bioline tubing released the least bubbles in high fluid level. At low level, the Gambro tubing showed the least bubbles at flows 50,400 mL/min, and the Fresenius M.C. tubing showed the least bubbles at flows 400,600 mL/min. High fluid level in the air trap reduced generation of micro bubbles compared to low level, as did lower fluid flow versus high flow. The design of the air trap was also of importance. [source] Periostin promotes a fibroblastic lineage pathway in atrioventricular valve progenitor cellsDEVELOPMENTAL DYNAMICS, Issue 5 2009Russell A. Norris Abstract Differentiation of prevalvular mesenchyme into valve fibroblasts is an integral step towards the development of functionally mature cardiac valves. Although clinically relevant, little is known regarding the molecular and cellular mechanisms by which this process proceeds. Genes that are regulated in a spatio-temporal pattern during valve remodeling are candidates for affecting this differentiation process. Based on its expression pattern, we have focused our studies on the role of the matricellular gene, periostin, in regulating the differentiation of cushion mesenchymal cells into valve fibroblasts. Herein, we demonstrate that periostin expression is coincident with and regulates type I collagen protein production, a major component of mature valve tissue. Adenoviral-mediated knock-down of periostin in atrioventricular mesenchyme resulted in a decrease in collagen I protein expression and aberrant induction of myocyte markers indicating an alteration in AV mesenchyme differentiation. In vitro analyses using a novel "cardiotube" assay further demonstrated that expression of periostin regulates lineage commitment of valve precursor cells. In these cells, expression of periostin and collagen I are regulated, in part, by TGF,-3. We further demonstrate that TGF,-3, through a periostin/collagen pathway, enhances the viscoelastic properties of AV cushion tissue surface tension and plays a crucial role in regulating valve remodeling. Thus, data presented here demonstrate that periostin, a TGF,-3 responsive gene, functions as a crucial mediator of chick AV valve maturation via promoting mesenchymal-to-fibroblast differentiation while blocking differentiation of alternative cell types (myocytes). Developmental Dynamics 238:1052,1063, 2009. © 2009 Wiley-Liss, Inc. [source] Effects of selected polybrominated diphenyl ether flame retard ants on lake trout (Salvelinus namaycush) thymocyte viability, apoptosis, and necrosisENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2005Kelly L. Birchmeier Abstract Polybrominated diphenyl ether (PBDE) flame-retardants have been identified as an emergent contaminants issue in many parts of the world. In vitro analyses were conducted to test the hypothesis that selected PBDEs congeners affect viability, apoptosis, and necrosis of thymocytes from laboratory-reared lake trout (Salvelinus namaycush). At current environmental levels (<1 mg/L), effects of the tested PBDEs on thymocytes were negligible. However, at 100 mg/L, major effects were seen for congener brominated diphenyl ether 47 (BDE-47) and minor effects were seen for congener BDE-99. [source] Aging-dependent generation of suppressive CD4+CD25,R123loCD103+ T,cells in miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2003Jun Shimizu Abstract Advancing age is associated with significant alterations in immune functions, including a decline in CD4 T,cell function, in both mice and humans. In our previous report, we showed that CD4+CD25, T,cells in aged (24-month-old) mice, especially after in vitro pre-stimulation of these cells, exhibit hyporesponsive and suppressive properties. We examined here whether the suppressive activity of aged CD4+CD25, T,cells is ascribable to a particular population within these cells. In vitro analyses revealed that cell populations rapidly extruding Rhodamine-123 (R123) (referred to as R123lo cells) in aged CD4+CD25, T,cells have a more potent suppressive function compared with R123hi populations. In addition, CD103+ cells in freshly prepared aged CD4+CD25,R123lo T,cells had a most potent suppressive activity. Both R123hi and R123lo populations had individually stronger suppressive activity after pre-stimulation than before pre-stimulation. Furthermore, the R123lo population in young CD4+CD25, T,cells also had different properties from R123hi T cells: low responsiveness, no additive effect in proliferation assays, and the gain of a suppressive function after in vitro pre-stimulation. Takentogether, these results suggest that CD4+CD25,R123lo T,cells are a unique population within whole CD4+CD25, T,cells. This population exists in the earlystage of the life span, and the properties in this population become obvious with aging, that is the gain of their suppressive activity. [source] Developmental changes in cellular and extracellular structural macromolecules in the secondary palate and in the nasal cavity of the mouseEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2010Forugh Vaziri Sani Vaziri Sani F, Kaartinen V, El Shahawy M, Linde A, Gritli-Linde A. Developmental changes in cellular and extracellular structural macromolecules in the secondary palate and nasal cavity of the mouse. Eur J Oral Sci 2010; 118: 221,236. © 2010 The Authors. Journal compilation© 2010 Eur J Oral Sci The aim of this study was to analyse the hitherto largely unknown expression patterns of some specific cellular and extracellular molecules during palate and nasal cavity development. We showed that epithelia of the developing palate and the vomerine epithelium express similar sets of structural proteins. With the exception of keratin 15, which becomes barely detectable in the elevated palatal shelves, nearly all of these proteins become upregulated at the presumptive areas of fusion and in the adhering epithelia of the palate and nasal septum. In vivo and in vitro analyses indicated that reduction in the amount of keratin 15 protein is independent of Tgf,,Alk5 signalling. Foxa1 expression also highlighted the regionalization of the palatal and nasal epithelia. Owing to the lack of reliable markers of the palatal periderm, the fate of peridermal cells has been controversial. We identified LewisX/stage-specific embryonic antigen-1 as a specific peridermal marker, and showed that numerous peridermal cells remain trapped in the medial epithelial seam (MES). The fate of these cells is probably apoptosis together with the rest of the MES cells, as we provided strong evidence for this event. Heparan sulphate, chondroitin-6-sulphate, and versican displayed dynamically changing distribution patterns. The hitherto-unknown innervation pattern of the developing palate was revealed. These findings may be of value for unravelling the pathogenesis of palatal clefting. [source] Dynamics of yeast prion aggregates in single living cellsGENES TO CELLS, Issue 9 2006Shigeko Kawai-Noma Prions are propagating proteins that are ordered protein aggregates, in which the phenotypic trait is retained in the altered protein conformers. To understand the dynamics of the prion aggregates in living cells, we directly monitored the fate of the aggregates using an on-chip single-cell cultivation system as well as fluorescence correlation spectroscopy (FCS). Single-cell imaging revealed that the visible foci of yeast prion Sup35 fused with GFP are dispersed throughout the cytoplasm during cell growth, but retain the prion phenotype. FCS showed that [PSI+] cells, irrespective of the presence of foci, contain diffuse oligomers, which are transmitted to their daughter cells. Single-cell observations of the oligomer-based transmission provide a link between previous in vivo and in vitro analyses of the prion and shed light on the relationship between the protein conformation and the phenotype. [source] Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK)GENES TO CELLS, Issue 2 2002Hidemasa Goto Background: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments. Results: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization. Conclusion: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation. [source] Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia,,HEPATOLOGY, Issue 5 2006Cara L. Mack Biliary atresia is an inflammatory fibrosclerosing lesion of the bile ducts that leads to biliary cirrhosis and is the most frequent indication for liver transplantation in children. The pathogenesis of biliary atresia is not known; one theory is that of a virus-induced, subsequent autoimmune-mediated injury of bile ducts. The aim of this study was to determine whether autoreactive T cells and autoantibodies specific to bile duct epithelia are present in the rotavirus (RRV)- induced murine model of biliary atresia and whether the T cells are sufficient to result in bile duct inflammation. In vitro analyses showed significant increases in IFN-,,producing T cells from RRV-diseased mice in response to bile duct epithelial autoantigen. Adoptive transfer of the T cells from RRV-diseased mice into naïve syngeneic SCID recipients resulted in bile duct,specific inflammation. This induction of bile duct pathology occurred in the absence of detectable virus, indicating a definite response to bile duct autoantigens. Furthermore, periductal immunoglobulin deposits and serum antibodies reactive to bile duct epithelial protein were detected in RRV-diseased mice. In conclusion, both cellular and humoral components of autoimmunity exist in murine biliary atresia, and the progressive bile duct injury is due in part to a bile duct epithelia,specific T cell,mediated immune response. The role of cellular and humoral autoimmunity in human biliary atresia and possible interventional strategies therefore should be the focus of future research. (HEPATOLOGY 2006;44:1231,1239.) [source] CASRdb: calcium-sensing receptor locus-specific database for mutations causing familial (benign) hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia,HUMAN MUTATION, Issue 2 2004Svetlana Pidasheva Abstract Familial hypocalciuric hypercalcemia (FHH) is caused by heterozygous loss-of-function mutations in the calcium-sensing receptor (CASR), in which the lifelong hypercalcemia is generally asymptomatic. Homozygous loss-of-function CASR mutations manifest as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism, which occur in infancy. Activating mutations in the CASR gene have been identified in several families with autosomal dominant hypocalcemia (ADH), autosomal dominant hypoparathyroidism, or hypocalcemic hypercalciuria. Individuals with ADH may have mild hypocalcemia and relatively few symptoms. However, in some cases seizures can occur, especially in younger patients, and these often happen during febrile episodes due to intercurrent infection. Thus far, 112 naturally-occurring mutations in the human CASR gene have been reported, of which 80 are unique and 32 are recurrent. To better understand the mutations causing defects in the CASR gene and to define specific regions relevant for ligand-receptor interaction and other receptor functions, the data on mutations were collected and the information was centralized in the CASRdb (www.casrdb.mcgill.ca), which is easily and quickly accessible by search engines for retrieval of specific information. The information can be searched by mutation, genotype,phenotype, clinical data, in vitro analyses, and authors of publications describing the mutations. CASRdb is regularly updated for new mutations and it also provides a mutation submission form to ensure up-to-date information. The home page of this database provides links to different web pages that are relevant to the CASR, as well as disease clinical pages, sequence of the CASR gene exons, and position of mutations in the CASR. The CASRdb will help researchers to better understand and analyze the mutations, and aid in structure,function analyses. Hum Mutat 24:107,111, 2004. © 2004 Wiley-Liss, Inc. [source] Differential Contribution of Osteoclast- and Osteoblast-Lineage Cells to CpG-Oligodeoxynucleotide (CpG-ODN) Modulation of Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2005Alla Amcheslavsky Abstract CpG-ODNs modulate osteoclast differentiation through Toll-like receptor 9 (TLR9). Using TLR9-deficient mice, we found that activation of TLR9 on both osteoclast precursors and osteoblasts mediate the osteoclastogenic effect of CpG-ODN. Osteoclastic TLR9 is more important for this activity. Introduction: Bacterial infections cause pathological bone loss by accelerating differentiation and activation of the osteoclast. A variety of bacteria-derived molecules have been shown to enhance osteoclast differentiation through activation of Toll-like receptors (TLRs). We have shown that CpG-oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA and exerting their cellular activities through TLR9, modulate osteoclast differentiation in a complex manner: the ODNs inhibit the activity of the physiological osteoclast differentiation factor RANKL in early osteoclast precursors (OCPs) but markedly stimulate osteoclastogenesis in cells primed by RANKL. Materials and Methods: Osteoclast precursors and osteoblasts from TLR9-deficient (TLR9,/,) and wildtype (TLR9+/+) mice were used for in vitro analyses of osteoclast differentiation and modulation of signal transduction and gene expression. Results: As expected CpG-ODN did not exert any activity in cells derived from TLR9,/,mice; these cells, however, responded in a normal manner to other stimuli. Using bone marrow/osteoblasts co-cultures from all possible combinations of TLR9,/, and TLR9+/+ mice-derived cells, we showed that TLR9 in the two lineages is required for CpG-ODN induction of osteoclastogenesis. Conclusions: CpG-ODN modulates osteoclastogenesis in a TLR9-dependent manner. Activation of TLR9 in bone marrow-derived osteoclasts precursors is more crucial to induction of osteoclastogenesis than activation of the osteoblastic TLR9. [source] Microbial induction of CARD15 expression in intestinal epithelial cells via toll-like receptor 5 triggers an antibacterial response loop,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006B. Begue With the discovery of CARD15 as susceptibility gene for Crohn's disease (CD) a first link to a potential defect in the innate immune system was made. In this work we aimed to analyze enterocyte NOD2/CARD15 expression and regulation in response to bacterial motifs and the consequences of the most common CD-specific CARD15 mutation on antibacterial responses of normal intestinal epithelial cells (IEC). Under normal conditions, IEC lines and ileal enterocytes did not express NOD2/CARD15 mRNA or protein, contrary to IEC derived from inflammatory CD sections. In vitro analyses revealed that the simple contact with non-pathogenic commensal E. Coli K12 was sufficient to induced NOD2/CARD15 mRNA and protein in human IEC (HIEC). We identified bacterial flagellin interacting with TLR5 as major motif in this regulation of NOD2/CARD15. E. Coli mutants not expressing flagellin (,FliC) failed to induce CARD15. Similarly, in HIEC transfected with a plasmid encoding dominant negative TLR5, no CARD15 induction was observed after K12 contact. Isolated TLR2 or TLR4 stimulation had no or only a marginal effect on NOD2/CARD15 expression. NOD2/CARD15 negative HIEC were unresponsive to muramyl dipeptide (MDP), but once NOD2/CARD15 was induced, HIEC and Caco2 cells responded to intra or extracellular MDP presentation with the activation of the NFkB pathway. IEC transfected with the Crohn-specific CARD15 mutant (F3020insC, FS) failed to activate NFkB after MDP-challenge, in contrast to CARD15WT IEC. In response to MDP, IEC induced a massive antibacterial peptide (ABP) response, seen in the apical release of CCL20. This was completely abolished in IEC carrying CARD15FS. These data suggest a critical role of NOD2/CARD15 in the bacterial clearance of the intestinal epithelium while CD-specific mutated NOD2/CARD15 causes an impaired epithelial barrier. J. Cell. Physiol. 209: 241,252, 2006. © 2006 Wiley-Liss, Inc. [source] Subtractive Screening for Probiotic Properties of Lactobacillus Species from the Human Gastrointestinal Tract in the Search for New ProbioticsJOURNAL OF FOOD SCIENCE, Issue 8 2007S. Delgado ABSTRACT:, In the search for new probiotics, 61 Lactobacillus spp. isolates, belonging to 12 species and isolated as dominant lactic acid bacteria from the feces of healthy humans, were subjected to a subtractive system of in vitro analyses, which included desirable and undesirable traits. Twenty-four isolates were able to grow in 2% bovine bile, of which 13 grew in acidified broth at pH 3.5 in acidified cysteine-containing MRS broth. Intrinsic resistance to certain antimicrobial agents (cefoxitin, metronidazole, vancomycin) was observed in most isolates, but atypical resistances to erythromycin, clindamycin, or tetracycline were also found in 5 strains. Undesirable traits such as ,-chymotrypsin or N-acetyl-,-glucosaminidase activities were not detected, but low ,-glucuronidase and moderate ,-glucosidase activities were recorded in 2 strains. Two Lactobacillus gasseri and 2 Lactobacillus paracasei selected strains inhibited several intestinal pathogens in an agar spot test, including strains of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus. They also adhered to human Caco-2 and HT-29 epithelial cells in a manner comparable to Lactobacillus rhamnosus strain GG, and were unable to degrade pig gastric mucin in a plate assay. Together, these results suggest these 4 strains to be good probiotic candidates, concluding that the subtractive screening devised in this work could be a valuable tool in large-scale surveys for probiotics. [source] Paracrine effect of transplanted rib chondrocyte spheroids supports formation of secondary cartilage repair tissue,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 9 2009Kolja Gelse Abstract The study's objective was to investigate if transplanted chondrocyte or periosteal cell spheroids have influence on ingrowing bone marrow-derived cells in a novel cartilage repair approach in miniature pigs. Autologous rib chondrocytes or periosteal cells were cultured as spheroids and press-fitted into cavities that were milled into large, superficial chondral lesions of the patellar joint surface. Within the milled cavities, the subchondral bone plate was either penetrated or left intact (full-thickness or partial-thickness cavities). The transplantation of chondrocyte spheroids into full-thickness cavities induced the formation of additional secondary repair cartilage that exceeded the original volume of the transplanted spheroids. The resulting continuous tissue was rich in proteoglycans and stained positive for type II collagen. Cell labeling revealed that secondarily invading repair cells did not originate from transplanted spheroids, but rather from arroded bone marrow. However, secondary invasion of repair cells was less pronounced following transplantation of periosteal cells and absent in partial-thickness cavities. According to in vitro analyses, these observations could be ascribed to the ability of chondrocyte spheroids to secrete relevant amounts of bone morphogenetic protein-2, which was not detected for periosteal cells. Transplanted chondrocyte spheroids exert a dual function: they provide cells for the repair tissue and have a stimulatory paracrine activity, which promotes ingrowth and chondrogenesis of bone marrow-derived cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Locally delivered rhTGF-,2 enhances bone ingrowth and bone regeneration at local and remote sites of skeletal injuryJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2001Dr. Sumner The purposes of the present study were to determine if recombinant human transforming growth factor-beta-2 (rhTGF-,2) enhances bone ingrowth into porous-coated implants and bone regeneration in gaps between the implant and surrounding host bone. The implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs in the presence of a 3-mm gap. In three treatment groups of animals, the test implant was treated with hydroxyapatite/tricalcium phosphate (HA/TCP) and rhTGF-,2 in buffer at a dose per implant of 1.2 ,g (n = 6), 12 ,g (n = 7), or 120 ,g (n = 7) and placed in the left humerus. In these same animals, an internal control implant treated only with HA/TCP and buffer was placed in the right humerus. In a non-TGF-, treated external control group of animals (n = 7), one implant was treated with HA/TCP while the contralateral implant was not treated with the ceramic. In vitro analyses showed that approximately 15% of the applied dose was released within 120 h with most of the release occurring in the first 24 h. The TGF-, treated implants had significantly more bone ingrowth than the controls with the greatest effect in the 12 ,g/implant group (a 2.2-fold increase over the paired internal control (P = 0.004) and a 4-fold increase over the external control (P < 0.001)). The TGF-, treated implants had significantly more bone formation in the gap than the controls with the greatest effect in the 12 and 120 ,g groups (1.8-fold increases over the paired internal controls (P = 0.003 and P = 0.012, respectively) and 2.8-fold increases over the external controls (P < 0.001 and P = 0.001, respectively)). Compared to the external controls, the internal control implants tended to have more bone ingrowth (1.9-fold increase, P = 0.066) and had significantly more bone formation in the gap (1.7-fold increase, P = 0.008). Thus, application of rhTGF-,2 to a porous-coated implant-stimulated local bone ingrowth and gap healing in a weakly dose-dependent manner and stimulated bone regeneration in the 3-mm gap surrounding the contralateral control implant, a site remote from the local treatment with the growth factor. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Conjugated linoleic acid evokes de-lipidation through the regulation of genes controlling lipid metabolism in adipose and liver tissueOBESITY REVIEWS, Issue 3 2005R. L. House Summary Conjugated linoleic acid (CLA) is a unique lipid that elicits dramatic reductions in adiposity in several animal models when included at ,,1% of the diet. Despite a flurry of investigations, the precise mechanisms by which conjugated linoleic acid elicits its dramatic effects in adipose tissue and liver are still largely unknown. In vivo and in vitro analyses of physiological modifications imparted by conjugated linoleic acid on protein and gene expression suggest that conjugated linoleic acid exerts its de-lipidating effects by modulating energy expenditure, apoptosis, fatty acid oxidation, lipolysis, stromal vascular cell differentiation and lipogenesis. The purpose of this review shall be to examine the recent advances and insights into conjugated linoleic acid's effects on obesity and lipid metabolism, specifically focused on changes in gene expression and physiology of liver and adipose tissue. [source] Immunological effects of stress in psoriasisBRITISH JOURNAL OF DERMATOLOGY, Issue 4 2009G. Schmid-Ott Summary Background, Psychological stress causes phenotypic changes in circulating lymphocytes and is regarded as an important trigger of the Th1-polarized inflammatory skin disease psoriasis. Objective, To study the effects of psychological stress on immunological parameters, i.e. membrane molecules relevant to the pathophysiology of psoriasis, especially cutaneous lymphocyte-associated antigens (CLA) involved in T and natural killer (NK) cells homing in on the skin. Methods, The severity of psoriasis was assessed in patients using the Psoriasis Area and Severity Index. Patients with psoriasis (n = 15) and healthy volunteers (n = 15) were exposed to brief psychological stress in the laboratory. In vitro analyses were conducted 1 h before, immediately following and 1 h after stress exposure. Peripheral T- and NK-cell subsets including CD8+ T lymphocytes, CLA+ lymphocytes and lymphocyte function-associated antigen type 1 (LFA-1)+ lymphocytes were analysed by flow cytometry. Results, We found a significant stress-induced increase of CD3+ T lymphocytes in patients with psoriasis only. Analyses of T-cell subsets revealed that this increase was observable for cytotoxic CD8+ T lymphocytes and CLA+ CD3+ lymphocytes. The total number of circulating NK cells (CD16+, CD56+) increased immediately after stress in both groups whereas only patients with psoriasis showed a significant increase in CLA+ NK cells. Conclusions, A higher stress-induced increase of CLA+ T and CLA+ NK cells in the circulation of patients with psoriasis might point to an increased ability of T and NK cells in the presence of psoriasis to home in on the skin during mental stress. Further studies are needed to verify these relationships in more detail and to investigate the time point at which these cells accumulate within lesional skin, and whether or not psychotherapy improves the quality of life of patients with psoriasis and influences stress-dependent parameters. [source] Protective role of the antidiabetic drug metformin against chronic experimental pulmonary hypertensionBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2009C Agard Background and purpose:, Pulmonary arterial hypertension (PAH) is associated with increased contraction and proliferation of pulmonary vascular smooth muscle cells. The anti-diabetic drug metformin has been shown to have relaxant and anti-proliferation properties. We thus examined the effect of metformin in PAH. Experimental approach:, Metformin effects were analysed in hypoxia- and monocrotaline-induced PAH in rats. Ex vivo and in vitro analyses were performed in lungs, pulmonary artery rings and cells. Key results:, In hypoxia- and monocrotaline-induced PAH, the changes in mean pulmonary arterial pressure and right heart hypertrophy were nearly normalized by metformin treatment (100 mg·kg,1·day,1). Pulmonary arterial remodelling occurring in both experimental models of PAH was also inhibited by metformin treatment. In rats with monocrotaline-induced PAH, treatment with metformin significantly increased survival. Metformin increased endothelial nitric oxide synthase phosphorylation and decreased Rho kinase activity in pulmonary artery from rats with PAH. These effects are associated with an improvement of carbachol-induced relaxation and reduction of phenylephrine-induced contraction of pulmonary artery. In addition, metformin inhibited mitogen-activated protein kinase activation and strongly reduced pulmonary arterial cell proliferation during PAH. In vitro, metformin directly inhibited pulmonary artery smooth muscle cell growth. Conclusions and implications:, Metformin protected against PAH, regardless of the initiating stimulus. This protective effect may be related to its anti-remodelling property involving improvement of endothelial function, vasodilatory and anti-proliferative actions. As metformin is currently prescribed to treat diabetic patients, assessment of its use as a therapy against PAH in humans should be easier. [source] FORMALIN-INDUCED INCREASE IN P2X3 RECEPTOR EXPRESSION IN DORSAL ROOT GANGLIA: IMPLICATIONS FOR NOCICEPTIONCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2009Ai-Hua Pan SUMMARY 1ATP-gated P2X receptors in nociceptive sensory neurons participate in the transmission of pain signals from the periphery to the spinal cord. The effect of formalin on the expression of P2X3 receptors in dorsal root ganglia (DRG) was characterized using molecular and immunological approaches and the patch-clamp technique. 2Adult Sprague-Dawley rats were injected with 100 µL of 5% formalin in the planar surface of the hindpaw and were killed 30 min and 1, 3, 6, 12, 24 and 48 h later for in vitro analyses. The expression and distribution of P2X3 receptors in the lumbar spinal cord and in L5/L6 DRG were examined; 24 and 48 h after formalin injection, currents in neurons were examined using whole-cell patch-clamp recording. 3Western blots showed that anti-P2X3 antibody recognized a major monomer of approximately 64 kDa in DRG. Immunoreactivity for P2X3 receptors was detected predominantly in the cytoplasm and plasma membrane of small (< 25 µm) and middle-sized (25,50 µm) DRG neurons. Expression of the P2X3 transcript in the DRG was unchanged 30 min and 1 h after formalin injection, but increased after 12 h. There was no distinct change in P2X3 immunostaining of the spinal cord lamina at 30 min or 1 h after injection, but after 24 h P2X3 labelling increased. At 24 h after the formalin injection, currents in isolated small and middle-sized DRG neurons were increased by 1 µmol/L ,,,-methylene-ATP. These currents were completely inhibited by 1 µmol/L A-317491, a potent and selective P2X3 receptor antagonist. 4These data suggest that formalin injection leads to early upregulation of P2X3 expression in the spinal cord and DRG and that this may be one of the mechanisms giving rise to nociception. [source] Balloon Debanding the Pulmonary Artery: In Vitro Studies and Early Clinical ExperienceCONGENITAL HEART DISEASE, Issue 4 2009Gareth J. Morgan MPhil ABSTRACT Despite increasing corrective procedures for children with congenital heart disease, there remains a place for surgical banding of the main pulmonary artery (PA). In the vast majority of cases, these bands eventually need to be removed. We examined three cases of percutaneous disruption of PA bands using balloon catheters at our institution. We also performed an in vitro study of PA band disruption mechanism and disruption pressure. Our in vitro study suggested a predictable burst pressure for PA bands over the range of diameters routinely used in pediatric practice. Of three patients who underwent interventional debanding, two patients had successful disruption of their PA bands with no reintervention at 19 months and 23 months follow up. Balloon disruption of surgical PA bands may offer a less invasive alternative to surgical band removal. In vitro analysis suggests that the burst pressure required and mechanism of disruption are predictable. [source] Repulsive guidance molecule/neogenin: a novel ligand-receptor system playing multiple roles in neural developmentDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2004Eiji Matsunaga The repulsive guidance molecule (RGM) is a membrane-bound protein originally isolated as an axon guidance molecule in the visual system. Recently, the transmembrane protein, neogenin, has been identified as the RGM receptor. In vitro analysis with retinal explants showed that RGM repels temporal retinal axons and collapses their growth cones through neogenin-mediated signaling. However, RGM and neogenin are also broadly expressed at the early embryonic stage, suggesting that they do not only control the guidance of visual axons. Gene expression perturbation experiments in chick embryos showed that neogenin induces cell death, and its ligand, RGM, blocks the pro-apoptotic activity of neogenin. Thus, RGM/neogenin is a novel dependence ligand/receptor couple as well as an axon guidance molecular complex. [source] In vivo and in vitro analysis of the vasculogenic potential of avian proepicardial and epicardial cells,DEVELOPMENTAL DYNAMICS, Issue 4 2006Juan A. Guadix Abstract Coronary vessel formation is a special case in the context of embryonic vascular development. A major part of the coronary cellular precursors (endothelial, smooth muscle, and fibroblastic cells) derive from the proepicardium and the epicardium in what can be regarded as a late event of angioblastic and smooth muscle cell differentiation. Thus, coronary morphogenesis is dependent on the epithelial,mesenchymal transformation of the proepicardium and the epicardium. In this study, we present several novel observations about the process of coronary vasculogenesis in avian embryos, namely: (1) The proepicardium displays a high vasculogenic potential, both in vivo (as shown by heterotopic transplants) and in vitro, which is modulated by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor signals; (2) Proepicardial and epicardial cells co-express receptors for platelet-derived growth factor-BB and VEGF; (3) Coronary angioblasts (found all through the epicardial, subepicardial, and compact myocardial layers) express the Wilms' tumor associated transcription factor and the retinoic acid-synthesizing enzyme retinaldehyde-dehydrogenase-2, two markers of the coelomic epithelium involved in coronary endothelium development. All these results contribute to the development of our knowledge on the vascular potential of proepicardial/epicardial cells, the existent interrelationships between the differentiating coronary cell lineages, and the molecular mechanisms involved in the regulation of coronary morphogenesis. Developmental Dynamics 235:1014,1026, 2006. © 2006 Wiley-Liss, Inc. [source] A human phospholamban promoter polymorphism in dilated cardiomyopathy alters transcriptional regulation by glucocorticoids,HUMAN MUTATION, Issue 5 2008Kobra Haghighi Abstract Depressed calcium handling by the sarcoplasmic reticulum (SR) Ca-ATPase and its regulator phospholamban (PLN) is a key characteristic of human and experimental heart failure. Accumulating evidence indicates that increases in the relative levels of PLN to Ca-ATPase in failing hearts and resulting inhibition of Ca sequestration during diastole, impairs contractility. Here, we identified a genetic variant in the PLN promoter region, which increases its expression and may serve as a genetic modifier in dilated cardiomyopathy (DCM). The variant AF177763.1:g.203A>C (at position ,36,bp relative to the PLN transcriptional start site) was found only in the heterozygous form in 1 out of 296 normal subjects and in 22 out of 381 cardiomyopathy patients (heart failure at age of 18,44 years, ejection fraction=22±9%). In vitro analysis, using luciferase as a reporter gene in rat neonatal cardiomyocytes, indicated that the PLN-variant increased activity by 24% compared to the wild type. Furthermore, the g.203A>C substitution altered the specific sequence of the steroid receptor for the glucocorticoid nuclear receptor (GR)/transcription factor in the PLN promoter, resulting in enhanced binding to the mutated DNA site. These findings suggest that the g.203A>C genetic variant in the human PLN promoter may contribute to depressed contractility and accelerate functional deterioration in heart failure. Hum Mutat 29(5), 640,647, 2008. © 2008 Wiley-Liss, Inc. [source] Mutations in RYR1 in malignant hyperthermia and central core disease,HUMAN MUTATION, Issue 10 2006Rachel Robinson Abstract The RYR1 gene encodes the skeletal muscle isoform ryanodine receptor and is fundamental to the process of excitation,contraction coupling and skeletal muscle calcium homeostasis. Mapping to chromosome 19q13.2, the gene comprises 106 exons and encodes a protein of 5,038 amino acids. Mutations in the gene have been found in association with several diseases: the pharmacogenetic disorder, malignant hyperthermia (MH); and three congenital myopathies, including central core disease (CCD), multiminicore disease (MmD), and in an isolated case of a congenital myopathy characterized on histology by cores and rods. The majority of gene mutations reported are missense changes identified in cases of MH and CCD. In vitro analysis has confirmed that alteration of normal calcium homeostasis is a functional consequence of some of these changes. Genotype,phenotype correlation studies performed using data from MH and CCD patients have also suggested that mutations may be associated with a range of disease severity phenotypes. This review aims to summarize the current understanding of RYR1 mutations reported in association with MH and CCD and the present viewpoint on the use of mutation data to aid clinical diagnosis of these conditions. Hum Mutat 27(10), 977,989, 2006. © 2006 Wiley-Liss, Inc. [source] Tumor expressed PTHrP facilitates prostate cancer-induced osteoblastic lesionsINTERNATIONAL JOURNAL OF CANCER, Issue 10 2008Jinhui Liao Abstract Expression of parathyroid hormone-related protein (PTHrP) correlates with prostate cancer skeletal progression; however, the impact of prostate cancer-derived PTHrP on the microenvironment and osteoblastic lesions in skeletal metastasis has not been completely elucidated. In this study, PTHrP overexpressing prostate cancer clones were stably established by transfection of full length rat PTHrP cDNA. Expression and secretion of PTHrP were verified by western blotting and IRMA assay. PTHrP overexpressing prostate cancer cells had higher growth rates in vitro, and generated larger tumors when inoculated subcutaneously into athymic mice. The impact of tumor-derived PTHrP on bone was investigated using a vossicle co-implant model. Histology revealed increased bone mass adjacent to PTHrP overexpressing tumor foci, with increased osteoblastogenesis, osteoclastogenesis and angiogenesis. In vitro analysis demonstrated pro-osteoclastic and pro-osteoblastic effects of PTHrP. PTHrP enhanced proliferation of bone marrow stromal cells and early osteoblast differentiation. PTHrP exerted a pro-angiogenic effect indirectly, as it increased angiogenesis but only in the presence of bone marrow stromal cells. These data suggest PTHrP plays a role in tumorigenesis in prostate cancer, and that PTHrP is a key mediator for communication and interactions between prostate cancer and the bone microenvironment. Prostate cancer-derived PTHrP is actively involved in osteoblastic skeletal progression. © 2008 Wiley-Liss, Inc. [source] Computational approach to the blood,aluminum problem?,INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 2 2007Christopher Exley Abstract Aluminum has no known function in biota and, when biologically available, is inimical to life. A key to understanding its potential toxicity in humans is its transport in blood. A consensus of opinion has identified the binding of aluminum by the iron-transport protein transferrin as the preeminent factor in the transport of aluminum in blood, although this idea has emanated from in vitro analysis of isolated blood and has never been tested in vivo. We have highlighted what we believe to be inadequacies of our present understanding of aluminum transport in blood, and we have proposed the application of computational methods to test rigorously what we have coined "the blood-aluminum problem." © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007 [source] Rietveld structure and in vitro analysis on the influence of magnesium in biphasic (hydroxyapatite and ,-tricalcium phosphate) mixturesJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2009S. Kannan Abstract The structure of two different Mg-substituted biphasic (HAP and ,-TCP) mixtures along with the biphasic mixtures without substituted Mg2+ was investigated using Rietveld refinement technique. The substituted Mg2+ was found in the ,-TCP phase and its influence on the composition has led to an increase in HAP content of Mg-containing biphasic mixtures when compared with the HAP content detected in pure biphasic mixtures. The refined structural parameters of Ca10(PO4)6(OH)2 and ,-Ca3(PO4)2 confirmed that all the investigated compositions have crystallized in the corresponding hexagonal (space group P63/m) and rhombohedral (space group R3c) structures. The substitution of lower sized magnesium was found preferentially incorporated at the sixfold-coordinated Ca (5) site of ,-TCP, which is due to the strong Ca (5)·O interaction among all the five different Ca sites of ,-Ca3(PO4)2. The in vitro tests using primary culture of osteoblasts showed that all the tested samples are biocompatible and promising materials for in vivo studies. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [source] In vitro analysis of intestinal absorption of cadmium and calcium in rainbow trout fed with calcium- and cadmium-supplemented dietsJOURNAL OF FISH BIOLOGY, Issue 3 2006B. Baldisserotto The protective effects of dietary Ca2+ supplementation against Cd accumulation in rainbow trout Oncorhynchus mykiss fed with Cd-contaminated food were evaluated in relation to chronic changes in intestinal absorption rates. The changes were measured ,in vitro'. The control diet contained c. 20 mg Ca2+ g,1 food and 0·25 ,g Cd g,1 food; the experimental diets were supplemented with CaCO3 and Cd(NO3)2·4H2O to levels of 50 mg Ca2+ g,1 food and 300 ,g Cd g,1 food, alone and in combination. The Ca2+ and Cd absorption rates were measured using radiotracers (45Ca, 109Cd) at total Ca2+ and Cd concentrations of 3·0 and 0·12 mmol l,1, respectively in the intestinal saline. Chronically elevated dietary Cd caused a significant increase in Cd absorption rate by up to 10-fold at 30 days in the mid-intestine. The high Ca2+ diet prevented this up-regulation of Cd transport rate. Conversely, intestinal Ca2+ absorption was significantly increased by two- to five-fold by the Ca2+ -supplemented diet at 30 days in both the mid- and posterior intestine, and this effect was eliminated when Cd was simultaneously elevated in the diet. Ca2+ and Cd probably interact at common pathways and transport mechanisms in the intestine, though independent pathways may also exist. [source] Synthesis and characterization of 4,6-dichloroindole-based radioligands for imaging the glycine site of the NMDA ion channelJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 2 2002R. N. Waterhouse Abstract To provide effective PET or SPECT ligands for the glycine binding site of the NMDA ion channel, we have synthesized and characterized in vitro four substituted derivatives of the potent glycine site antagonist 3-[2-[(phenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxylic acid (Ki=3.0 nM). These new ligands contain groups amenable to labeling with C-11 for PET, or I-123 for SPECT. In vitro analysis of these compounds revealed that placement of a methoxy group at either the ortho or para position of the phenylaminocarbonyl group significantly reduced receptor affinity (Ki=74.0±8.1 and 26.5±4.9 nM, respectively), as did placement of an iodine at the para position (Ki=60.4±8.2 nM). However, the meta -methoxy derivative (4b) maintained high affinity (Ki=4.8±0.9 nM) for the glycine site and was therefore labeled with carbon-11 by reacting the corresponding desmethyl derivative with [11C]methyl iodide. Radiochemical yields of 14±10% (EOS), and high specific activity (1.2±0.5 Ci/,mol (EOS, n=7)) were realized, and the product was prepared in a sterile saline solution suitable for in vivo use. Copyright © 2002 John Wiley & Sons, Ltd. [source] In vitro analysis of synergism and antagonism of different nucleoside/nucleotide analogue combinations on the inhibition of human immunodeficiency virus type 1 replication,JOURNAL OF MEDICAL VIROLOGY, Issue 2 2009M. Perez-Olmeda Abstract In this study we have developed an in vitro system to evaluate the combined effect of two NRTIs on HIV replication and to assess their antagonism or synergy. Synergy or antagonism effect was determined in peripheral blood mononuclear cells (PBMCs) to approach a more physiological model than T-cell lines. PBMCs were infected with a full-length HIV-1 clone carrying the luciferase gene as a reporter. The following combinations were investigated: zidovudine+stavudine (ZDV,+, d4T), lamivudine,+,abacavir (3TC,+,ABC), lamivudine,+,didanosine (3TC,+,ddI), lamivudine,+ stavudine (3TC,+,d4T), tenofovir,+,stavudine (TDF,+,d4T), tenofovir,+,didanosine (TDF,+,ddI), tenofovir,+,abacavir (TDF,+,ABC), tenofovir,+, lamivudine (TDF,+,3TC), tenofovir,+,zidovudine (TDF,+,ZDV), stavudine,+,didanosine (d4T,+,ddI), zidovudine,+,lamivudine (ZDV,+,3TC), abacavir,+, didanosine (ABC,+,ddI), zidovudine,+,didanosine (ZDV,+,ddI), and abacavir,+,stavudine (ABC,+, d4T). The effect of combining two drugs was evaluated with a quantitative method based on the median-effect principle of Chou and Talalay. A synergistic effect was observed with combinations containing TDF and ZDV or d4T, d4T and ddI and ZDV plus 3TC. In contrast, combinations including TDF,+,ddI, 3TC,+,ddI, ABC,+,ddI, and ZDV,+,ddI showed an antagonistic effect on the inhibition of viral replication at all levels of inhibition tested. Lower antagonistic effect was also found in drug combinations that included 3TC,+,ABC, 3TC,+,TDF, 3TC,+,d4T, and TDF,+, ABC. In conclusion, the method developed allows to measure in vitro the effect of different combinations of two NRTIs on HIV replication. The results suggest that combined therapy including TDF with thymidine analogues may be considered for future therapeutic options in contrast to clearly antagonistic combinations such us TDF plus ddI or 3TC plus ddI, that would explain virological failure in clinical studies when these combinations were used. J. Med. Virol. 81:211,216, 2009. © 2008 Wiley-Liss, Inc. [source] Successful treatment of an entecavir-resistant hepatitis B virus variantJOURNAL OF MEDICAL VIROLOGY, Issue 12 2007Hiromi Yatsuji Abstract Emergence of a lamivudine (LAM)-resistant hepatitis B virus (HBV) with amino acid substitutions in the YMDD motif is a well-documented problem during long-term LAM therapy. Entecavir (ETV) is a new drug approved for treatment of HBV infection with or without LAM-resistant mutants. This report describes an ETV-resistant strain of HBV, which emerged after prolonged ETV therapy in a patient who did not respond to LAM therapy. Direct sequence analysis of the ETV-resistant strain showed appearance of amino acid substitution rtS202G in the reverse transcriptase (RT) domain, together with rtL180M,+,M204V substitution that had developed at the emergence of LAM-resistant mutant. In vitro analysis demonstrated that the rtL180M,+,M204V,+,S202G mutant strain displayed a 200-fold and a 5-fold reduction in susceptibility to ETV compared with the wild- type and the rtL180M,+,M204V mutant strain, respectively. Adefovir was effective against the ETV-resistant strain both in vitro and during the clinical course. In conclusion, this study showed that virological and biochemical breakthrough due to ETV could occur in patients infected with LAM-resistant HBV and confirmed that the addition of rtS202G substitution to the rtL180M,+,M204V mutant strain is responsible for ETV resistance and we could treat the resistant mutant successfully. J. Med. Virol. 79:1811,1817, 2007. © 2007 Wiley-Liss, Inc. 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